Isoprenoids derived from mevalonic acid are required for a variety of cell biological functions including cell proliferation, membrane structure and function, cell adhesion and cytoskeletal functions. A subset of cellular proteins (including members of the ras oncogene- and G-protein families) is modified by covalent attachment of long chain isoprenoids (farnesyl, geranylgeranyl) to cysteines located in CAAX boxes close to the carboxyterminus. The functions of isoprenylation are not well understood. Farnesylation is thought to serve as a signal for other covalent modifications of ras-like proteins and nuclear lamin B, which increase their hydrophobicity and facilitate interaction of these proteins with membranes. Moreover, farnesylation is obligatory for the transforming activity of ras oncogenes. Much remains to be learned about isoprenylation in epithelial cells, which is the primary focus of this project. We are using MDCK and other renal adenocarcinoma cell lines to investigate the requirement for isoprenylation in the regulation of cell shape and adhesion. In addition, we are developing permeabilized cell models capable of incorporating substrates and inhibitors of isoprenylation that are poorly taken up by intact cells. We have found that renal adenocarcinoma cells incorporate 3H-mevalonate into proteins ranging in MW from greater than 130 Kd to 17 KD. Cell lines differ in their mevalonate requirements for growth and viability, but in all cells examined depletion of cellular mevalonate results in changes in cell-cell adhesive contacts and tubulin organization. The underlying biochemical mechanisms are being investigated. Eduardo Sainz , a chemist in my laboratory, has developed HPLC and TLC methods to separate isoprenoids from other cellular lipids and an assay for protein prenyltransferase activity in cell free extracts. Initial studies on cell permeabilization suggest a novel role for ATP in cell adhesion, which is currently under investigation.